U.S. patent application number 10/505983 was filed with the patent office on 2006-05-11 for piperidine ouracil used as a medicament for treating bacterial infections.
Invention is credited to Michael Brands, Kerstin Ehlert, Dietmar Flubacher, Jorg Keldenich, Alexander Kuhl, Christoph Ladel, Michael Otteneder, Niels Svenstrup.
Application Number | 20060100224 10/505983 |
Document ID | / |
Family ID | 27674993 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060100224 |
Kind Code |
A1 |
Svenstrup; Niels ; et
al. |
May 11, 2006 |
Piperidine ouracil used as a medicament for treating bacterial
infections
Abstract
The invention relates to piperidine ouracil and a method for the
production thereof in addition to the use thereof in the production
of medicaments for treating and/or in the prophylaxis of diseases,
especially bacterial diseases. ##STR1##
Inventors: |
Svenstrup; Niels;
(Wuppertal, DE) ; Kuhl; Alexander; (Hagen, DE)
; Flubacher; Dietmar; (Freiburg, DE) ; Brands;
Michael; (Hamden, CT) ; Ehlert; Kerstin;
(Velbert, DE) ; Ladel; Christoph; (Ivrea, IT)
; Otteneder; Michael; (Arlesheim, CH) ; Keldenich;
Jorg; (Wuppertal, DE) |
Correspondence
Address: |
JEFFREY M. GREENMAN
BAYER PHARMACEUTICALS CORPORATION
400 MORGAN LANE
WEST HAVEN
CT
06516
US
|
Family ID: |
27674993 |
Appl. No.: |
10/505983 |
Filed: |
February 13, 2003 |
PCT Filed: |
February 13, 2003 |
PCT NO: |
PCT/EP03/01405 |
371 Date: |
December 23, 2005 |
Current U.S.
Class: |
514/269 ;
544/310 |
Current CPC
Class: |
C07D 409/14 20130101;
C07D 413/14 20130101; C07D 417/14 20130101; C07D 405/14 20130101;
C07D 401/14 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/269 ;
544/310 |
International
Class: |
A61K 31/513 20060101
A61K031/513; C07D 403/02 20060101 C07D403/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 2002 |
DE |
102 08 256.1 |
Claims
1. A compound of the formula ##STR73## in which R.sup.1 is
heteroaryl, where heteroaryl may be substituted by 0, 1, 2 or 3
substituents selected independently of one another from the group
consisting of alkyl, alkoxy, alkylthio, halogen, alkanoyl, hydroxy,
trifluoromethyl, trifluoromethoxy, nitro, amino, alkylamino,
alkanoylamino, cyano, carboxy, cycloalkyl, heterocyclyl, aryl and
optionally methyl-substituted heteroaryl, R.sup.2 is hydrogen or
alkyl, R.sup.3 is a substituent of the following formula ##STR74##
in which R.sup.3-1 and R.sup.3-2 are selected independently of one
another from the group consisting of alkyl, alkenyl, alkynyl,
alkylthio, cycloalkyl and halogen, or R.sup.3-1 and R.sup.3-2 form
together with the carbon atoms to which they are bonded a
cycloalkyl or heterocyclyl ring which is optionally substituted by
up to 3 halogen.
2. A compound of the formula (I) as claimed in claim 1, in which
the uracil ring is linked via positions 3, 4 or 5 to the piperidine
ring, R.sup.1 is heteroaryl, where heteroaryl may be substituted by
0, 1, 2 or 3 substituents selected independently of one another
from the group consisting of alkyl, alkoxy, alkylthio, halogen,
hydroxy, carboxy, heterocyclyl and aryl, R.sup.2 is hydrogen or
alkyl, R.sup.3 is a substituent of the following formula ##STR75##
in which R.sup.3-1 and R.sup.3-2 are selected independently of one
another from the group consisting of C.sub.1-C.sub.6-alkyl,
C.sub.2-C.sub.6-alkenyl, C.sub.2-C.sub.6-alkynyl,
C.sub.1-C.sub.6-alkylthio, C.sub.3-C.sub.6-cycloalkyl and halogen,
or R.sup.3-1 and R.sup.3-2 form together with the carbon atoms to
which they are bonded a C.sub.3-C.sub.6-Cycloalkyl or heterocyclyl
ring which is optionally substituted by up to 3 halogen.
3. A compound of the formula (I) as claimed in claim 1 or 2, in
which the uracil ring is linked via positions 3, 4 or 5 to the
piperidine ring, R.sup.1 is heteroaryl, where heteroaryl may be
substituted by 0, 1 or 2 substituents selected independently of one
another from the group consisting of alkyl, halogen and carboxy,
R.sup.2 is hydrogen, and R.sup.3 is a substituent of the following
formula ##STR76## in which R.sup.3-1 and R.sup.3-2 are selected
independently of one another from the group consisting of methyl,
ethyl, fluorine and chlorine, or R.sup.3-1 and R.sup.3-2 form
together with the carbon atoms to which they are bonded a
C.sub.5-cycloalkyl ring which is optionally substituted by up to 2
substituents selected independently of one another from the group
consisting of chlorine or fluorine.
4. A compound of the formula (I) as claimed in claim 1 or 2, in
which the uracil ring is linked via positions 3, 4 or 5 to the
piperidine ring.
5. A compound of the formula (I) as claimed in any of claims 1 to
4, in which R.sup.1 is a 5- to 6-membered heteroaryl radical.
6. A compound of the formula (I) as claimed in claim 1, 2, 4 or 5,
in which R.sup.2 is hydrogen.
7. A compound of the formula (I) as claimed in any of claims 1 to
6, in which R.sup.3 is selected from the group of ##STR77##
8. A process for preparing compounds of the formula (I) by reacting
compounds of the formula ##STR78## in which R.sup.2 and R.sup.3
have the meaning indicated in claim 1, with compounds of the
general formula ##STR79## in which R.sup.1 has the meaning
indicated in claim 1, and X.sup.1 is halogen, preferably chlorine
or bromine, or hydroxy.
9. A compound as claimed in any of claims 1 to 7 for the treatment
and/or prophylaxis of diseases.
10. A medicament comprising at least one compound as claimed in any
of claims 1 to 7 in combination with at least one pharmaceutically
suitable, pharmaceutically acceptable carrier or excipient.
11. The use of compounds as claimed in any of claims 1 to 7 for
producing a medicament for the treatment and/or prophylaxis of
bacterial infections.
12. A medicament as claimed in claim 10 for the treatment and/or
prophylaxis of bacterial infections.
13. A method for controlling bacterial infections in humans and
animals by administering an antibacterially effective amount of at
least one compound as claimed in any of claims 1 to 7.
Description
[0001] The invention relates to piperidinouracils and process for
their preparation, and to their use for producing medicaments for
the treatment and/or prophylaxis of diseases, especially of
bacterial diseases.
[0002] Gram-positive eubacteria contain three different DNA
polymerase exonucleases which are referred to as Pol 1, Pol 2 and
Pol 3. Pol 3 is an enzyme which is necessary for the replicative
synthesis of DNA.
[0003] The suitability of uracil derivatives for the treatment of
bacterial infections has already been known for some time. Thus, WO
01/29010 describes 3-aminocarbonyl-substituted phenylaminouracils,
WO 96/06614 describes 3-alkylidene-substituted uracils, WO 00/71523
describes 3-alkanoyloxyalkyluracils and WO 00/20556 describes
uracils with zinc finger-active unit as antibacterial
compounds.
[0004] J. Med. Chem., 1999, 42, 2035, Antimicro. Agents and
Chemotherapy, 1999, 43, 1982 and Antimicro. Agents and
Chemotherapy, 2000, 44, 2217 describe phenylaminouracils as
antibacterial compounds.
[0005] One object of the present invention is to provide novel,
alternative soluble compounds with antibacterial effect for the
treatment of bacterial diseases in humans and animals.
[0006] The present invention relates to compounds of the formula
(I), ##STR2## in which
[0007] R.sup.1 is heteroaryl, [0008] where heteroaryl may be
substituted by 0, 1, 2 or 3 substituents selected independently of
one another from the group consisting of alkyl, alkoxy, alkylthio,
halogen, alkanoyl, hydroxy, trifluoromethyl, trifluoromethoxy,
nitro, amino, alkylamino, alkanoylamino, cyano, carboxy,
cycloalkyl, heterocyclyl, aryl and optionally methyl-substituted
heteroaryl,
[0009] R.sup.2 is hydrogen or alkyl,
[0010] R.sup.3 is a substituent of the following formula ##STR3##
[0011] in which [0012] R.sup.3-1 and R.sup.3-2 are selected
independently of one another from the group consisting of alkyl,
alkenyl, alkynyl, alkylthio, cycloalkyl and halogen, or [0013]
R.sup.3-1 and R.sup.3-2 form together with the carbon atoms to
which they are bonded a cycloalkyl or heterocyclyl ring which is
optionally substituted by up to 3 halogen.
[0014] The compounds of the invention may also be in the form of
their salts, solvates or solvates of the salts.
[0015] The compounds of the invention may, depending on their
structure, exist in stereoisomeric forms (enantiomers,
diastereomers). The invention therefore relates to the enantiomers
or diastereomers and respective mixtures thereof. The
stereoisomerically pure constituents can be isolated in a known
manner from such mixtures of enantiomers and/or diastereomers.
[0016] The invention also relates, depending on the structure of
the compounds, to tautomers of the compounds.
[0017] Salts preferred for the purposes of the invention are
physiologically acceptable salts of the compounds of the
invention.
[0018] Physiologically acceptable salts of the compounds (I)
include acid addition salts of mineral acids, carboxylic acids and
sulfonic acids, e.g. salts of hydrochloric acid, hydrobromic acid,
sulfuric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid,
naphthalenedisulfonic acid, acetic acid, propionic acid, lactic
acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic
acid and benzoic acid.
[0019] Physiologically acceptable salts of the compounds (I) also
include salts of conventional bases, such as by way of example and
preferably alkali metal salts (e.g. sodium and potassium salts),
alkaline earth metal salts (e.g. calcium and magnesium salts) and
ammonium salts derived from ammonia or organic amines having 1 to
16 C atoms, such as by way of example and preferably ethylamine,
diethylamine, triethylamine, ethyldiisopropylamine,
monoethanolamine, diethanolamine, triethanolamine,
dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine,
N-methylmorpholine, dihydroabiethylamine, arginine, lysine,
ethylenediamine and methylpiperidine.
[0020] Solvates refers for the purposes of the invention to those
forms of the compounds which form a complex in the solid or liquid
state through coordination with solvent molecules. Hydrates are a
specific form of solvates in which the coordination takes place
with water.
[0021] For the purposes of the present invention, the substituents
have the following meaning unless specified otherwise:
[0022] Alkyl per se and "alk" and "alkyl" in alkoxy, alkylamino
alkylthio, alkanoyl and alkanoylamino stand for a linear or
branched alkyl radical having ordinarily from 1 to 6, preferably 1
to 4, particularly preferably 1 to 3, carbon atoms, by way of
example and preferably methyl, ethyl, n-propyl, isopropyl,
tert-butyl, n-pentyl and n-hexyl.
[0023] Alkenyl stands for a straight-chain or branched alkenyl
radical having 2 to 6 carbon atoms. Preference is given to a
straight-chain or branched alkenyl radical having 2 to 4,
particularly preferably having 2 to 3 carbon atoms. Preferred
examples which may be mentioned are: vinyl, allyl, n-prop-1-en-1-yl
and n-but-2-en-1-yl.
[0024] Alkynyl stands for a straight-chain or branched alkenyl
radical having 2 to 6 carbon atoms. Preference is given to a
straight-chain or branched alkenyl radical having 2 to 4,
particularly preferably having 2 to 3 carbon atoms. Preferred
examples which may be mentioned are: n-prop-1-yn-1-yl and
n-but-2-yn-1-yl.
[0025] Alkoxy stands by way of example and preferably for methoxy,
ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and
n-hexoxy.
[0026] Alkylamino stands for an alkylamino radical having one or
two alkyl substituents (chosen independently of one another),, by
way of example and preferably methylamino, ethylamino,
n-propylamino, isopropylamino, tert-butylamino, n-pentylamino,
n-hexylamino, N,N-dimethylamino, N,N-diethylamino,
N-ethyl-N-methylamino, N-methyl-N-n-propylamino,
N-isopropyl-N-n-propylamino, N-t-butyl-N-methylamino,
N-ethyl-N-n-pentylamino and N-n-hexyl-N-methylamino.
[0027] Alkylthio stands by way of example and preferably for
methylthio, ethylthio, n-propylthio, isopropylthio, tert-butylthio,
n-pentylthio and n-hexylthio.
[0028] Alkanoyl stands by way of example and preferably for acetyl
and ethylcarbonoyl.
[0029] Alkanoylamino stands by way of example and preferably for
acetylamino and ethylcarbonylamino.
[0030] Cycloalkyl stands for a cycloalkyl group having ordinarily 3
to 8, preferably 5 to 7, carbon atoms, by way of example and
preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl.
[0031] Aryl stands for a mono- to tricyclic aromatic, carbocyclic
radical ordinarily having from 6 to 14 carbon atoms; by way of
example and preferably phenyl, naphthyl and phenanthrenyl.
[0032] Heteroaryl stands for an aromatic, mono- or bicyclic radical
ordinarily having from 5 to 10, preferably 5 to 6 ring atoms and up
to 5, preferably up to 4, in particular up to 3, heteroatoms from
the series S, O and N, by way of example and preferably thienyl,
furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, imidazolyl,
pyridyl, pyrimidyl, pyridazinyl, indolyl, indazolyl, benzofuranyl,
benzothiophenyl, quinolinyl, isoquinolinyl.
[0033] Heterocyclyl stands for a mono- or polycyclic, preferably
mono- or bicyclic, nonaromatic heterocyclic radical ordinarily
having from 4 to 10, preferably 5 to 8, in particular 5 or 6, ring
atoms and up to 3, preferably up to 2, heteroatoms and/or hetero
groups from the series N, O, S, SO, SO.sub.2. The heterocyclyl
radicals may be saturated or partially unsaturated. Preference is
given to 5- to 6-membered, monocyclic saturated heterocyclyl
radicals having up to two heteroatoms from the series O, N and S,
such as by way of example and preferably tetrahydrofuran-2-yl,
pyrrolidin-2-yl, pyrrolidin-3-yl, pyrrolinyl, piperidinyl,
morpholinyl, perhydroazepinyl.
[0034] Halogen stands for fluorine, chlorine, bromine and
iodine.
[0035] A symbol * on a bond denotes the point of linkage in the
molecule.
[0036] If radicals in the compounds of the invention are
substituted, the radicals may, unless otherwise specified, have one
or more identical or different substituents. Substitution by up to
three identical or different substituents is preferred.
Substitution by one substituent is very particularly preferred.
[0037] Preference is given for the purposes of the present
invention to compounds of the general formula (I)
in which
[0038] the uracil ring is linked via positions 3, 4 or 5 to the
piperidine ring,
[0039] R.sup.1 is heteroaryl, [0040] where heteroaryl may be
substituted by 0, 1, 2 or 3 substituents selected independently of
one another from the group consisting of alkyl, alkoxy, alkylthio,
halogen, hydroxy, carboxy, heterocyclyl and aryl,
[0041] R.sup.2 is hydrogen or alkyl,
[0042] R.sup.3 is a substituent of the following formula ##STR4##
[0043] in which [0044] R.sup.3-1 and R.sup.3-2 are selected
independently of one another from the group consisting of
C.sub.1-C.sub.6-alkyl, C.sub.2-C.sub.6-alkenyl,
C.sub.2-C.sub.6-alkynyl, C.sub.1-C.sub.6-alkylthio,
C.sub.3-C.sub.6-cycloalkyl and halogen, or [0045] R.sup.3-1 and
R.sup.3-2 form together with the carbon atoms to which they are
bonded a C.sub.3-C.sub.6-cycloalkyl or heterocyclyl ring which is
optionally substituted by up to 3 halogen.
[0046] Preference is given for the purposes of the present
invention to compounds of the general formula (I),
in which
[0047] the uracil ring is linked via positions 3, 4 or 5 to the
piperidine ring,
[0048] R.sup.1 is heteroaryl, [0049] where heteroaryl may be
substituted by 0, 1, or 2 substituents selected independently of
one another from the group consisting of alkyl, halogen and
carboxy,
[0050] R.sup.2 is hydrogen, and
[0051] R.sup.3 is a substituent of the following formula ##STR5##
[0052] in which [0053] R.sup.3-1 and R.sup.3-2 are selected
independently of one another from the group consisting of methyl,
ethyl, fluorine and chlorine, or [0054] R.sup.3-1 and R.sup.3-2
form together with the carbon atoms to which they are bonded a
C.sub.5-cycloalkyl ring which is optionally substituted by up to 2
substituents selected independently of one another from the group
consisting of chlorine or fluorine.
[0055] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which the
uracil ring is linked via positions 3, 4 or 5 to the piperidine
ring.
[0056] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which R.sup.1
is a 5- to 6-membered heteroaryl radical, in particular a
5-membered heteroaryl radical.
[0057] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which R.sup.1
is isoxazolyl or furyl.
[0058] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which R.sup.2
is hydrogen.
[0059] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which
[0060] R.sup.3 is selected from the group of ##STR6##
[0061] Preference is given for the purposes of the present
invention among these in particular to compounds of the general
formula (I) in which
[0062] R.sup.3 is selected from the group of ##STR7##
[0063] Preference is also given for the purposes of the present
invention to compounds of the general formula (I) in which R.sup.3
is selected from the group of ##STR8##
[0064] The present invention further relates to a process for
preparing the compounds of the general formula (I), where compounds
of the general formula ##STR9## in which
[0065] R.sup.2 and R.sup.3 have the meaning indicated above, are
reacted with compounds of the general formula ##STR10## in
which
[0066] R.sup.1 has the meaning indicated above, and
[0067] X.sup.1 is halogen, preferably chlorine or bromine, or
hydroxy.
[0068] In the case where X.sup.1 is halogen, the reaction takes
place in inert solvents, if appropriate in the presence of a base,
preferably in a temperature range from 0.degree. C. to 50.degree.
C. under atmospheric pressure.
[0069] Examples of inert solvents are halohydrocarbons such as
methylene chloride, trichloromethane, tetrachloromethane,
trichloroethane, tetrachloroethane, 1,2-dichloroethane or
trichloroethylene, ethers such as diethyl ether, methyl tert-butyl
ether, dioxane, tetrahydrofuran, glycol dimethyl ether or
diethylene glycol dimethyl ether, hydrocarbons such as benzene,
xylene, toluene, hexane, cyclohexane or petroleum fractions, or
other solvents such as nitromethane, ethyl acetate, acetone,
dimethylformamide, dimethylacetamide, 1,2-dimethoxyethane,
2-butanone, dimethyl sulfoxide, acetonitrile or pyridine, with
preference for dioxane or methylene chloride.
[0070] Examples of bases are alkali metal hydroxides such as sodium
or potassium hydroxide, or alkali metal carbonates such as cesium
carbonate, sodium or potassium carbonate, or amides such as lithium
diisopropylamide, or other bases such as DBU, triethylamine or
diisopropylethylamine, preferably diisopropylethylamine or
triethylamine.
[0071] In the case where X.sup.1 is hydroxy, the reaction takes
place in inert solvents in the presence of conventional condensing
agents, where appropriate in the presence of a base, preferably in
a temperature range of from room temperature to 50.degree. C. under
atmospheric pressure.
[0072] Examples of inert solvents are halohydrocarbons such as
methylene chloride, trichloromethane, tetrachloromethane,
trichloroethane, tetrachloroethane, 1,2-dichloroethane or
trichloroethylene, ethers such as diethyl ether, methyl tert-butyl
ether, dioxane, tetrahydrofuran, glycol dimethyl ether or
diethylene glycol dimethyl ether, hydrocarbons such as benzene,
xylene, toluene, hexane, cyclohexane or petroleum fractions, or
other solvents such as nitromethane, ethyl acetate, acetone,
dimethylformamide, dimethylacetamide, 1,2-dimethoxyethane, dimethyl
sulfoxide, acetonitrile or pyridine, with preference for
tetrahydrofuran, dimethylformamide, 1,2-dichloroethane or methylene
chloride.
[0073] Examples of conventional condensing agents are carbodiimides
such as, for example, N,N'-diethyl-, N,N'-dipropyl-,
N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide,
N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene
(PS-carbodiimide) or carbonyl compounds such as
carbonyldiimidazole, or 1,2-oxazolium compounds such as
2-ethyl-5-phenyl-1,2-oxazolium-3-sulfate or
2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino
compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline,
or propanephosphonic anhydride, or isobutyl chloroformate, or
bis(2-oxo-3-oxaolidinyl)phosphoryl chloride or
benzotriazolyloxytri(dimethylamino)phosphonium hexafluorophosphate,
or O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU),
2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU) or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt), or
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP), or mixtures thereof.
[0074] Examples of bases are alkali metal carbonates such as, for
example, sodium or potassium carbonate, or sodium or potassium
bicarbonate, or organic bases such as trialkylamines, e.g.
triethylamine, N-methylmorpholine, N-methylpiperidine,
4-dimethylaminopyridine or diisopropylethylamine.
[0075] The combination of
N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC), 1-hydroxybenzotriazole (HOBt) and triethylamine in
dimethylformamide, or
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) and diisopropylethylamine in
tetrahydrofuran, is particularly preferred.
[0076] The compounds of the formula (III) are known or can be
prepared in analogy to known processes.
[0077] One process for preparing compounds of the general formula
(II) is characterized in that compounds of the formula ##STR11## in
which
[0078] R.sup.2 has the meaning indicated above, [0079] are reacted
with compounds of the general formula H.sub.2N--R.sup.3 (V) in
which
[0080] R.sup.3 has the meaning indicated above, [0081] where
appropriate in the presence of a base.
[0082] The reaction takes place where appropriate in inert
solvents, preferably in a temperature range from 100.degree. C. to
150.degree. C. under atmospheric pressure.
[0083] Examples of inert solvents are halohydrocarbons such as
ethers such as dioxane, glycol dimethyl ether or diethylene glycol
dimethyl ether, hydrocarbons such as benzene, xylene or toluene, or
other solvents dimethylformamide, dimethylacetamide, dimethyl
sulfoxide, pyridine or 1-methylpyrrolidinone, with preference for
dioxane or 1-methylpyrrolidinone.
[0084] Examples of bases are alkali metal hydroxides such as sodium
or potassium hydroxide, or alkali metal carbonates such as cesium
carbonate, sodium or potassium carbonate, or amides such as lithium
diisopropylamide, or other bases such as DBU, triethylamine or
diisopropylethylamine, preferably diisopropylethylamine or
triethylamine.
[0085] The compounds of the formula (V) are known or can be
prepared in analogy to known processes.
[0086] One process for preparing the compounds of the general
formula (IV) is characterized in that compounds of the formula
##STR12## in which
[0087] R.sup.2 has the meaning indicated above,
are reacted with trifluoroacetic acid, where appropriate in the
presence of methylene chloride, preferably in a temperature range
from 60.degree. C. to reflux of the solvent under atmospheric
pressure.
[0088] One process for preparing the compounds of the general
formula (VI) is characterized in that the compound of the formula
##STR13## with compounds of the general formula ##STR14## in
which
[0089] R.sup.2 has the meaning indicated above,
under Mitsunobu conditions in the presence of a phosphine and of a
dialkyl azodicarboxylate, preferably in the presence of
triphenylphosphine and diethyl azodicarboxylate, in tetrahydrofuran
at 0.degree. C. to room temperature under atmospheric pressure.
[0090] The compounds of the formula (VII) and (VIII) are known or
can be prepared in analogy to known processes.
[0091] The preparation routes are to be illustrated by way of
example by the following diagrams:
Preparation of alkylpiperidinyl-6-chlorouracils
[0092] ##STR15##
Preparation of deprotected alkylpiperidinyl-6-chlorouracils
[0093] ##STR16##
Preparation of 6-aminoaryl-alkylpiperidinyluracils
[0094] ##STR17##
Preparation of 6-aminoaryl-alkyl-N'-acylpiperidinyluracils
[0095] ##STR18##
[0096] The compounds of the invention show a valuable range of
pharmaceutical and pharmacokinetic effects which could not have
been predicted. They are therefore suitable for use as medicaments
for the treatment and/or prophylaxis of diseases in humans and
animals.
[0097] The compounds of the invention are particularly effective
against bacteria and bacteroid microorganisms, especially
Gram-positive bacteria. They are therefore particularly well-suited
for the prophylaxis and chemotherapy of local and, where
appropriate, systemic infections caused by these pathogens in human
and veterinary medicine.
[0098] It is possible for example to treat and/or prevent local
and/or systemic diseases caused by the following pathogens or by
mixtures of the following pathogens:
[0099] Gram-positive cocci, e.g. staphylococci (Staph. aureus,
Staph. epidermidis) and streptococci (Strept. agalactiae, Strept.
faecalis, Strept. pneumoniae, Strept. pyogenes), and strictly
anaerobic bacteria such as, for example, clostridium, also
mycoplasmas (M. pneumoniae, M. hominis, M. urealyticum).
[0100] The above list of pathogens is merely by way of example and
by no means to be regarded as restrictive. Examples of diseases
which are caused by the mentioned pathogens or mixed infections,
and can be prevented, improved or cured by the compounds of the
invention, and which may be mentioned are:
[0101] Infectious diseases in humans such as, for example, septic
infections, bone and joint infections, skin infections,
postoperative wound infections, abscesses, phlegmon, wound
infections, infected burns, burn wounds, infections in the oral
region, infections after dental operations, septic arthritis,
mastitis, tonsillitis, genital infections and eye infections.
[0102] Apart from humans, it is also possible to treat bacterial
infections in other species. Examples which may be mentioned
are:
[0103] Pig: sepsis, metritis-mastitis-agalactiae syndrome,
mastitis;
[0104] Ruminants (cattle, sheep, goats): sepsis, bronchopneumonia,
mycoplasmosis, genital infections;
[0105] Horse: bronchopneumonias, puerperal and postpuerperal
infections;
[0106] Dogs and cats: bronchopneumonia, dermatitis, otitis, urinary
tract infections, prostatitis;
[0107] Poultry (chickens, turkeys, quail, pigeons, ornamental birds
and others): mycoplasmosis, chronic airway diseases,
psittacosis.
[0108] It is likewise possible to treat bacterial diseases in the
rearing and management of productive and ornamental fish, in which
case the antibacterial spectrum is extended beyond the pathogens
mentioned above to further pathogens such as, for example,
brucella, campylobacter, listeria, erysipelothris, nocardia.
[0109] The active ingredient may have systemic and/or local
effects. For this purpose, it can be administered in a suitable
manner such as, for example, by the oral, parenteral, pulmonary,
nasal, sublingual, lingual, buccal, rectal, transdermal,
conjunctival or otic route, or as implant. Parenteral
administration is preferred.
[0110] The active ingredient can be administered in suitable
administration forms for these administration routes.
[0111] Administration forms suitable for oral administration are
known ones which deliver the active ingredient in a rapid and/or
modified way, such as, for example, tablets (uncoated and coated
tablets, e.g. tablets provided with coatings resistant to gastric
juice), capsules, sugar-coated tablets, granules, pellets, powders,
emulsions, suspensions and solutions.
[0112] Parenteral administration can take place with avoidance of
an absorption step (intravenous, intraarterial, intracardiac,
intraspinal or intralumbar) or with inclusion of an absorption
(intramuscular, subcutaneous, intracutaneous, percutaneous or
intraperitoneal). Administration forms suitable for parenteral
administration include preparations for injection and infusion in
the form of solutions, suspensions, emulsions, lyophilisates and
sterile powders. Intravenous administration is preferred.
[0113] Examples suitable for the other administration routes are
medicinal forms for inhalation (inter alia powder inhalers,
nebulizers), nasal drops/solutions, sprays; tablets for lingual,
sublingual or buccal administration, or capsules, suppositories,
preparations for the eyes and ears, vaginal capsules, aqueous
suspensions (lotions, shaking mixtures), lipophilic suspensions,
ointments, creams, milk, pastes, dusting powders or implants.
[0114] The active ingredients can be converted in a manner known
per se into the administration forms listed. This takes place with
use of inert nontoxic, pharmaceutically suitable excipients. These
include inter alia carriers (e.g. microcrystalline cellulose),
solvents (e.g. liquid polyethylene glycols), emulsifiers (e.g.
sodium dodecyl sulfate), dispersants (e.g. polyvinylpyrrolidone),
synthetic and natural biopolymers (e.g. albumin), stabilizers (e.g.
antioxidants such as ascorbic acid), colorants (e.g. inorganic
pigments such as iron oxides) or masking tastes and/or odors.
[0115] It has generally proved advantageous for parenteral
administration to administer amounts of about 0.001 to 10 mg/kg,
preferably about 0.01 to 1 mg/kg, of body weight to achieve
effective results. On oral administration the amount is about 0.01
to 500 mg/kg, preferably about 1 to 10 mg/kg, of body weight.
[0116] It may nevertheless be necessary where appropriate to
deviate from the amounts mentioned, in particular as a function of
the body weight, administration route, individual response to the
active ingredient, type of preparation and time or interval over
which administration takes place.
[0117] Particular preference is given to parenteral, especially
intravenous, administration, e.g. as iv bolus injection (i.e. as
single dose, e.g. by syringe), short infusion (i.e. infusion over a
period of up to one hour) or long infusion (i.e. infusion over a
period of more than one hour). The administered volume may in these
cases be, depending on the specific conditions, between 0.5 to 30,
in particular 1 to 20 ml on iv bolus injection, between 25 to 500,
in particular 50 to 250 ml on short infusion and between 50 to
1000, in particular 100 to 500 ml on long infusion. It may for this
purpose be advantageous for the active ingredient to be provided in
solid form (e.g. as lyophilisate) to be dissolved in the dissolving
medium only directly before administration.
[0118] (It is necessary in these cases for the administration forms
to be sterile and pyrogen-free. They may be based on aqueous or
mixtures of aqueous and organic solvents.
[0119] These include, for example, aqueous solutions, mixtures of
aqueous and organic solvents (especially ethanol, polyethylene
glycol (PEG) 300 or 400), aqueous solutions containing
cyclodextrins or aqueous solutions containing emulsifiers
(surface-active solubilizers, e.g. lecithin or Pluronic F 68,
Solutol HS15, Cremophor). Aqueous solutions are preferred in this
connection.
[0120] Formulations suitable for parenteral administration are
those which are substantially isotonic and euhydric, e.g. those
with a pH between 3 and 11, in particular 6 and 8, especially
around 7.4.
[0121] Solutions for injection are packaged in suitable containers
made of glass or plastic, e.g. in vials. These may have a volume of
from 1 to 1000, in particular 5 to 50 ml. The solution can be
removed directly therefrom and administered. In the case of a
lyophilisate, it is dissolved in the vial by injecting a suitable
solvent and is then removed.
[0122] Solutions for infusion are packaged in suitable containers
made of glass or plastic, e.g. in bottles or collapsible plastic
bags. These may have a volume of from 1 to 1000, in particular 50
to 500, ml.
[0123] The percentage data in the following tests and examples are,
unless indicated otherwise, percentages by weight; parts are parts
by weight. Solvent ratios, dilution ratios and concentration data
for liquid/liquid solutions are in each case based on volume.
A. EXAMPLES
Abbreviations:
[0124] aq. aqueous [0125] Bn benzyl [0126] BOP
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate [0127] DMSO dimethyl sulfoxide [0128] DMF
dimethylformamide [0129] eq. equivalent [0130] ESI electrospray
ionization (in MS) [0131] h hour [0132] HPLC high pressure, high
performance liquid chromatography [0133] LC-MS coupled liquid
chromatography-mass spectroscopy [0134] MS mass spectroscopy [0135]
NMR nuclear magnetic resonance spectroscopy [0136] Pd/C
palladium/carbon [0137] RP-HPLC reverse phase HPLC [0138] RT room
temperature [0139] R.sub.t retention time (in HPLC) HPLC and LC-MS
Methods: Method 1: Instrument Micromass Quattro LCZ
[0140] Column: Symmetry C18, 50 mm.times.2.1 mm, 3.5 .mu.m;
temperature: 40.degree. C.; flow rate=0.5 mlmin.sup.-1;
eluent-A=CH.sub.3CN+0.1% formic acid, eluent B=water+0.1% formic
acid; gradient 0.0 min 10% A.fwdarw.4 min 90% A.fwdarw.6 min 90%
A
Method 2: Instrument Micromass Platform LCZ
[0141] Column: Symmetry C18, 50 mm.times.2.1 mm, 3.5 .mu.m;
temperature: 40.degree. C.; flow rate=0.5 mlmin.sup.-1; eluent
A=CH.sub.3CN+0.1% formic acid, eluent B=water+0.1% formic acid;
gradient 0.0 min 10% A.fwdarw.4 min 90% A.fwdarw.6 min 90% A
Method 3: Instrument: Finnigan MAT 900S, TSP: P4000, AS3000,
UV3000HR
[0142] Column: symmetry C 18, 150 mm.times.2.1 mm, 5.0 .mu.m;
eluent C: water, eluent B: water+0.3 g of 35% strength HCl, eluent
A: acetonitrile; gradient: 0.0 min 2% A.fwdarw.2.5 min 95%
A.fwdarw.5 min 95% A; oven: 70.degree. C.; flow rate: 1.2 mlmin; UV
detection: 210 nm
Starting Compounds:
Example I
1-[(Benzyloxy)methyl]-6-chloro-2,4(1H,3H)pyrimidinedione
[0143] ##STR19##
[0144] 45 g (307.09 mmol) of chlorouracil are mixed with 600. ml of
dimethylformamide and cooled to 0.degree. C. 3.22 g (405.46 mmol)
of lithium hydride are cautiously added (highly exothermic),
followed by stirring for 10 minutes. Then, 62.25 g (317.17 mmol) of
benzyl chloromethyl ether are added dropwise over the course of 10
minutes. This is followed by stirring at 0.degree. C. for 90
minutes. The reaction solution is mixed with 1000 ml of 2% strength
sodium hydroxide solution and extracted with 1000 ml of toluene.
The toluene phase is washed once with 100 ml of 2% strength sodium
hydroxide solution. The combined aqueous phases are adjusted to pH
3 with IN sulfuric acid. The precipitated solid is filtered off and
dried under high vacuum. 57.1 g (70% of theory) of product are
obtained.
[0145] .sup.1H-NMR (200 MHz, CDCl3): .delta.=4.69 (s, 2H), 5.54 (s,
2H), 5.87 (s, 1H), 7.35 (m, 5H), 8.45 (s, 1H).
Example II
tert-Butyl 4-(hydroxymethyl)-1-piperidinecarboxylate
[0146] ##STR20##
[0147] 12.1 g (105.06 mmol) of 4-(hydroxymethyl)piperidine are
dissolved together with 0.31 g (2.5 mmol) of
4-dimethylaminopyridine and 32.22 ml (231.13 mmol) of triethylamine
in 305 ml of dichloromethane. The mixture is cooled to 0.degree.
C., and 24.07 g (110.31 mmol) of di-tert-butyl pyrocarbonate are
added in portions. This is followed by stirring at room temperature
for 50 hours. The reaction solution is mixed with 25 ml of water.
The phases are separated, and the organic phase is washed once with
100 ml of 1M hydrochloric acid and once with 50 ml of sodium
chloride solution. It is dried over sodium sulfate and concentrated
in vacuo. 20 g (92% of theory) of product are obtained.
[0148] LC-MS (method 3): R.sub.t=2.13 min
[0149] MS (ESIpos): m/z=216 (M+H).sup.+
Example III
tert-Butyl
4-{[3-[(benzyloxy)methyl]-4-chloro-2,6-dioxo-3,6-dihydro-1(2H)--
pyrimidinyl]methyl}-1-piperidinecarboxylate
[0150] ##STR21##
[0151] 5 g (23.22 mmol) of the compound from example I, 6.19 g
(23.22 mmol) of the compound from example II and 6.7 g (25.55 mmol)
of triphenylphosphine are mixed in 153 ml of tetrahydrofuran. The
mixture is cooled to 0.degree. C., and 4.02 ml (25.55 mmol) of
diethyl azodicarboxylate are added dropwise over the course of 10
minutes. This is followed by stirring at room temperature for 72
hours. The reaction solution is concentrated in vacuo and purified
on silica gel 60.
[0152] 7.43 g (69% of theory) of product are obtained.
[0153] LC-MS (method 2): R.sub.t=4.7 min
[0154] MS (ESIpos): m/z=464 (M+H).sup.+
Example IV
6-Chloro-3-(4-piperidinylmethyl)-2,4(1H,3H)pyrimidinedione
[0155] ##STR22##
[0156] 9.85 g (21.23 mmol) of the compound from-example III are
mixed with 161 ml of trifluoroacetic acid and heated to reflux.
After 2 hours, the reaction solution is cooled and concentrated in
vacuo. It is taken up once more in methanol and again concentrated
in vacuo. The residue is recrystallized from 30 ml of ethyl
acetate. 5.43 g (99% of theory) of product are obtained.
[0157] LC-MS (method 1): R.sub.t=0.32 min
[0158] MS (ESIpos): m/z=244 (M+H).sup.+
Example V
6-(2,3-Dihydro-1H-inden-5-ylamino)-3-(4-piperidinylmethyl)-2,4(1H,3H)pyrim-
idinedione
[0159] ##STR23##
[0160] 0.77 g (3.18 mmol) of the compound from example IV are mixed
with 0.93 g (7 mmol) of 5-aminoindane and heated to 150.degree. C.
After 2 hours, the reaction solution is cooled. 10 ml of
dichloromethane and 5 ml of methanol are added, and this mixture is
added with stirring to 50 ml of diethyl ether. The precipitate is
filtered off and dried under high vacuum.
[0161] 0.667 g (61% of theory) of product is obtained.
[0162] LC-MS (method 1): R.sub.t=2.37 min
[0163] MS (ESIpos): m/z=341 (M+H).sup.+
Example VI
6-[(3-Ethyl-4-methylphenyl)amino]-3-(4-piperidinylmethyl)-2,4(1H,3H)pyrimi-
dinedione
[0164] ##STR24##
[0165] Preparation takes place as for example V from 1 g (4.104
mmol) of the compound from example IV, 0.845 g (4.924 mmol) of
3-ethyl-4-methylaniline hydrochloride and 1.57 ml (9.02 mmol) of
N,N-diisopropylethylamine.
[0166] 0.498 g (35% of theory) of product is obtained.
[0167] LC-MS (method 1): R.sub.t=2.47 min
[0168] MS (ESIpos): m/z=343 (M+1H).sup.+
PREPARATION EXAMPLES
Example 1
6-(2,3-Dihydro-1H-inden-5-ylamino)-3-({1-[(5-methyl-4-isoxazolyl)carbonyl]-
-4-piperidinyl}methyl)-2,4(1H,3H)pyrimidinedione
[0169] ##STR25##
[0170] 133.9 mg (0.39 mmol) of the compound from example V are
suspended together with 50 mg (0.39 mmol) of
5-methylisoxazole-4-carboxylic acid and 225.19 mg (0.43 mmol) of
1-benzotriazolyloxytripyrrolidinephosphonium hexafluorophosphate in
1.5 ml of tetrahydrofuran. 55.93 mg (0.43 mmol) of
N,N-diisopropylethylamine are added, followed by stirring at room
temperature for 24 hours. 0.5 ml of water is added to the reaction
solution. After 15 minutes, it is filtered through an
Extrelut/silica gel cartridge. 3 ml of methanol/dichloromethane in
the ratio 2:1 are used for washing. The filtrate is concentrated in
vacuo. The residue is taken up in ethyl acetate and washed twice
with sodium bicarbonate solution. Sodium sulfate is used for
drying, and the solution is concentrated in vacuo. Preparative HPLC
is used for purification.
[0171] 65 mg (37% of theory) of product are obtained.
[0172] LC-MS (method 2): R.sub.t=3.57 min
[0173] MS (ESIpos): m/z=450 (M+H).sup.+
[0174] The examples listed in the table below can be prepared from
the appropriate starting compounds in analogy to the methods
described above. TABLE-US-00001 Example Structure Analytical data 2
##STR26## LC-MS (method 2): Rt = 3.43 min MS (ESIpos): m/z = 450 (M
+ H) 3 ##STR27## LC-MS (method 2): Rt = 3.39 min MS (ESIpos): m/z =
446 (M + H) 4 ##STR28## LC-MS (method 1): Rt = 3.41 min MS
(ESIpos): m/z = 436 (M + H) 5 ##STR29## LC-MS (method 1): Rt = 3.48
min MS (ESIpos): m/z = 537 (M + H) 6 ##STR30## LC-MS (method 1): Rt
= 3.5 min MS (ESIpos): m/z = 435 (M + H) 7 ##STR31## LC-MS (method
1): Rt = 2.61 min MS (ESIpos): m/z = 435 (M + H) 8 ##STR32## LC-MS
(method 1): Rt = 3.63 min MS (ESIpos): m/z = 451 (M + H) 9
##STR33## LC-MS (method 1): Rt = 3.51 min MS (ESIpos): m/z = 438 (M
+ H) 10 ##STR34## LC-MS (method 2): Rt = 3.13 min MS (ESIpos): m/z
436 (M + H) 11 ##STR35## LC-MS (method 2): Rt = 3.5 min MS
(ESIpos): m/z = 502 (M + H) 12 ##STR36## LC-MS (method 2): Rt =
3.33 min MS (ESIpos): m/z = 449 (M + H) 13 ##STR37## LC-MS (method
2): Rt = 3.45 min MS (ESIpos): m/z = 453 (M + H) 14 ##STR38## LC-MS
(method 2): Rt = 3.21 min MS (ESIpos): m/z = 435 (M + H) 15
##STR39## LC-MS (method 2): Rt = 3.49 min MS (ESIpos): m/z = 450 (M
+ H) 16 ##STR40## LC-MS (method 2): Rt = 3.97 min MS (ESIpos): m/z
= 506 (M + H) 17 ##STR41## LC-MS (method 2): Rt = 3.13 min MS
(ESIpos): m/z = 436 (M + H) 18 ##STR42## LC-MS (method 2): Rt =
3.49 min MS (ESIpos): m/z = 502 (M + H) 19 ##STR43## LC-MS (method
2): Rt = 3.21 min MS (ESIpos): m/z = 435 (M + H) 20 ##STR44## LC-MS
(method 1): Rt = 3.48 min MS (ESIpos): m/z = 455 (M + H) 21
##STR45## LC-MS (method 2): Rt = 3.79 min MS (ESIpos): m/z = 550 (M
+ H) 22 ##STR46## 23 ##STR47## LC-MS (method 1): Rt = 3.85 min MS
(ESIpos): m/z = 506 (M + H) 24 ##STR48## LC-MS (method 2): Rt =
3.69 min MS (ESIpos): m/z = 448 (M + H) 25 ##STR49## LC-MS (method
1): Rt = 3.41 min MS (ESIpos): m/z = 453 (M + H) 26 ##STR50## LC-MS
(method 1): Rt = 3.16 min MS (ESIpos): m/z = 446 (M + H) 27
##STR51## LC-MS (method 1): Rt = 3.29 min MS (ESIpos): m/z = 446 (M
+ H) 28 ##STR52## LC-MS (method 2): Rt = 2.71 min MS (ESipos): m/z
= 435 (M + H) 29 ##STR53## LC-MS (method 1): Rt = 3.05 min MS
(ESIpos): m/z = 446 (M + H) 30 ##STR54## LC-MS (method 1): Rt =
3.65 min MS (ESIpos): m/z = 451 (M + H) 31 ##STR55## LC-MS (method
1): Rt = 3.45 min MS (ESIpos): m/z = 436 (M + H) 32 ##STR56## LC-MS
(method 1): Rt = 3.37 min MS (ESIpos): m/z = 450 (M + H) 33
##STR57## LC-MS (method 1): Rt = 3.85 min MS (ESIpos): m/z = 495 (M
+ H) 34 ##STR58## LC-MS (method 1): Rt = 3.57 min MS (ESIpos): m/z
= 467 (M + H) 35 ##STR59## LC-MS (method 1): Rt = 3.72 min MS
(ESIpos): m/z = 449 (M + H) 36 ##STR60## LC-MS (method 1): Rt =
3.84 min MS (ESIpos): m/z = 465 (M + H) 37 ##STR61## LC-MS (method
1): Rt = 3.51 min MS (ESIpos): m/z = 479 (M + H) 38 ##STR62## LC-MS
(method 2): Rt = 3.95 min MS (ESIpos): m/z = 544 (M + H) 39
##STR63## LC-MS (method 2): Rt = 3.74 min MS (ESIpos): m/z = 449 (M
+ H) 40 ##STR64## LC-MS (method 2): Rt = 3.86 min MS (ESIpos): m/z
= 465 (M + H) 41 ##STR65## LC-MS (method 1): Rt = 3.26 min MS
(ESIpos): m/z = 451 (M + H) 42 ##STR66## LC-MS (method 1): Rt =
3.59 min MS (ESIpos): m/z = 438 (M + H) 43 ##STR67## LC-MS (method
1): Rt = 3.29 min MS (ESIpos): m/z = 448 (M + H) 44 ##STR68## LC-MS
(method 1): Rt = 3.27 min MS (ESIpos): m/z = 437 (M + H) 45
##STR69## LC-MS (method 1): Rt = 3.52 min MS (ESIpos): m/z = 479 (M
+ H) 46 ##STR70## LC-MS (method 2): Rt = 4.11 min MS (ESIpos): m/z
= 526 (M + H) 47 ##STR71## LC-MS (method 1): Rt = 3.68 min MS
(ESIpos): m/z = 550 (M + H) 48 ##STR72## LC-MS (method 1): Rt =
3.78 min MS (ESIpos): m/z = 451 (M + H)
B. ASSESSMENT OF THE PHARMACOLOGICAL ACTIVITY
In Vitro Effect
[0175] The in vitro effect of the compounds of the invention can be
shown in the following assays:
Cloning, Expression and Purification of Pol III from S. aureus
[0176] To clone polC with an N-terminal His tag, the structural
gene polC is amplified from S. aureus genomic DNA with the aid of
PCR. The primers SAPol 31 5'-GCGCCATATGGACAGAGCAACAAAAATTTAA-3' and
SAPolrev 5'-GCGCGGATCCTTACATATCAAATATCGAAA-3' are used to introduce
the NdeI and BamHI restriction cleavage sites respectively in front
of and behind the amplified gene. After the PCR product which is
4300 bp in size has been digested with NdeI and BamHI, it is
ligated into the vector pET15b (Novagen, USA), which has likewise
been digested with NdeI and BamHI, and transformed into E. coli
XL-1 Blue.
[0177] After transformation into E. coli BL21(DE3), the cells are
cultivated for expression of PolC at 30.degree. C. in LB medium
with 100 .mu.g/ml ampicillin until the OD.sub.595 nm is 0.5, cooled
to 18.degree. C. and, after addition of 1 mM IPTG, incubated for a
further 20 hours. The cells are harvested by centrifugation, washed
once in PBS with 1 mM PMSF and taken up in 50 mM NaH.sub.2PO.sub.4
pH 8.0, 10 mM imidazole, 2 mM .beta.-mercaptoethanol, 1 mM PMSF,
20% glycerol. The cells are disrupted using a French press at
12,000 psi, the cell detritus is removed by centrifugation (27,000
TNR.times.g, 120 min, 4.degree. C.) and the supernatant is stirred
with an appropriate amount of Ni-NTA-agarose (from Quiagen,
Germany) at 4.degree. C. for 1 hour. After the gel matrix has been
packed into a column it is washed with 50 mM NaH.sub.2PO.sub.4 pH
8.0, 2 mM .beta.-mercaptoethanol, 20 mM imidazole, 10% glycerol,
and the purified protein is then eluted with the same buffer
containing 100 mM imidazole. The purified protein is mixed with 50%
glycerol and stored at -20.degree. C.
Determination of the IC.sub.50 for Polymerase III
[0178] The activity of PolC is measured in an enzymatically coupled
reaction, with the pyrophosphate formed during the polymerization
being converted with the aid of ATP sulfurylase into ATP, which is
detected using firefly luciferase. The reaction mixture contains,
in a final volume of 50 .mu.l, 50 mM Tris/Cl pH 7.5; 5 mM DTT, 10
mM MgCl.sub.2, 30 mM NaCl, 0.1 mg/ml BSA, 10% glycerol, 20 .mu.M
each dATP, dTTP, dCTP, 2U/ml activated calf thymus DNA (from
Worthington, USA), 20 .mu.M APS and 0.06 mM luciferin. The reaction
is started by adding purified PolC in a final concentration of
.about.2 nM and is incubated at 30.degree. C. for 30 min. The
amount of pyrophosphate formed is then converted into ATP by adding
ATP sulfurylase (Sigma, USA) in a final concentration of 5 nM and
incubating at 30.degree. C. for 15 min. After addition of 0.2 nM
firefly luciferase, the luminescence is measured in a luminometer
for 60 s. The IC.sub.50 is reported as the concentration of an
inhibitor which leads to 50% inhibition of the enzymic activity of
PolC. TABLE-US-00002 TABLE A Example No. IC.sub.50 (.mu.M) 9 0.14
13 0.09 17 0.34 21 0.11 26 0.22 39 0.33
Determination of the Minimum Inhibitory Concentrations (MIC) for a
Number of Microbes
[0179] The MIC values for various bacterial strains (S. aureus, S.
pneumoniae, E. faecalis) are carried out using the microdilution
method in BHI broth. The bacterial strains are cultured overnight
in BHI broth (staphylococci) or BHI broth+10% bovine serum
(streptococci, enterococci). The test substances are tested in a
concentration range from 0.5 to 256 .mu.g/ml. After serial dilution
of the test substances, the microtiter plates are inoculated with
the test microbes. The microbe concentration is about
1.times.10.sup.6 microbes/ml of suspension. The plates are
incubated at 37.degree. C. under 8% CO.sub.2 (for streptococci,
enterococci) for 20 h. The MIC is recorded as the lowest
concentration at which visible growth of the bacteria is completely
inhibited.
In Vivo Effect
[0180] The suitability of the compounds of the invention for
treating bacterial infections can be shown in the following animal
models:
Systemic Infection with S. aureus 133
[0181] S. aureus 133 cells are cultured overnight in BH broth. The
overnight culture is diluted 1:100 in fresh BH broth and amplified
for 3 hours. The bacteria, which are in the logarithmic phase of
growth, are spun down and washed 2.times. with buffered
physiological saline (303). Then a cell suspension is adjusted in a
photometer (model LP 2W from Dr. Lange, Germany) to an extinction
of 50 units in 303. After a dilution step (1:15), this suspension
is mixed 1:1 with a 10% strength mucin suspension. 0.25 ml of this
infection solution is administered ip per 20 g mouse. This
corresponds to a cell count of approximately 1.times.10E.sup.6
microbes/mouse. The ip therapy takes place 30 minutes after the
infection. Female CFW1 mice are used for the infection experiment.
The survival of the animals is recorded for 6 days.
C. EXEMPLARY EMBODIMENTS OF PHARMACEUTICAL COMPOSITIONS
[0182] The substances of the invention can be converted into
pharmaceutical preparations in the following ways:
Tablet:
Composition:
[0183] 100 mg of the compound of example 1, 50 mg of lactose
(monohydrate), 50 mg of corn starch (native), 10 mg of
polyvinylpyrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and
2 mg of magnesium stearate.
[0184] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12
mm.
Production:
[0185] A mixture of active ingredient, lactose and starch is
granulated with a 5% strength solution (m/m) of the PVP in water.
The granules are dried and then mixed with the magnesium stearate
for 5 min. This mixture is compressed using a conventional tablet
press (see above for format of the tablet). A compressive force of
15 kN is used as guideline for the compression.
Suspension Which Can Be Administered Orally:
Composition:
[0186] 1000 mg of the compound of example 1, 1000 mg of ethanol
(96%), 400 mg of Rhodigel (xanthan gum from FMC, Pennsylvania, USA)
and 99 g of water. 10 ml of oral suspension correspond to a single
dose of 100 mg of the compound of the invention.
Production:
[0187] The Rhodigel is suspended in ethanol, and the active
ingredient is added to the suspension. The water is added while
stirring. The mixture is stirred for about 6 h until the swelling
of the Rhodigel is complete.
Solution Which Can Be Administered Intravenously:
Composition:
[0188] 1 mg of the compound of example 1, 15 g of polyethylene
glycol 400 and 250 g of water for injections.
Production:
[0189] The compound of example 1 is dissolved together with
polyethylene glycol 400 in the water with stirring. The solution is
sterilized by filtration (pore diameter 0.22 .mu.m) and used to
fill heat-sterilized infusion bottles under aseptic conditions.
These are closed with infusion stoppers and crimped caps.
* * * * *